Effects of IL-6 and IL-11 on differentiation of cord blood CD34+ cells towards megakaryocytes

AIM: To investigate the effects of interleukin-6 (IL-6) and interleukin-11 (IL-11) on differentiation of cord blood CD34+ cells towards megakaryocytes and platelet production in vitro.METHODS: The CD34+ cells from fresh umbilical cord blood samples were cultured in serum-free culture medium with thrombopoietin (TPO) 50 μg/L,IL-3 10 μg/L,stem cell factor (SCF) 50 μg/L as control groups,then 10 μg/L IL-6 or IL-11 or IL-6＋IL-11 respectively was added as treatment groups.Mononuclear cells (MNCs) in cultured cells were detected by cell counter,megakaryocytes (CD41+ cells) and platelets were measured by flow cytometry,respectively.Platelet agglutination after thrombin induced was observed by microscopy and flow cytometry.RESULTS: Compared with the control group,the number of MNCs was not significantly different（P>0.05）,but the numbers of CD41+ cells and platelets were increased significantly (P<0.05) in treatment groups.There were more platelet particles in treatment groups than those in control group by microscopy and the results also showed that the cytoplasmic fragments from the cultures responded to thrombin induction.CONCLUSION: It is concluded that both IL-6 and IL-11 induce the cord blood CD34+ cells to differentiate towards megakaryocytes and produce platelets.     

Supportive effects of human aorta-gonad-mesonephero stromal cells on the directed differentiation of embryonic stem cells into hematopoietic stem cells

AIM: To direct embryonic stem cells (ESCs) into hematopoietic stem cells (HSCs) in vitro by simulating the hematogenic microenvironment in human early embryonic aorta-gonad-mesonephero (AGM) region.METHODS: Murine E14 embronic stem cell line was used for two-step differentiation.In the first step of primary differentiation,E14 ESCs were seeded into semisolid methylcellulose-based medium containing bone morphogenesis protein 4 (BMP4) and vascular endothelial growth factor (VEGF) for embryoid body (EB) formation.On days 3,6,9,12 and 15,single EB cells were analyzed for Flk-1+ cells amount through flow cytometry.In the second step,single cell from EB containing most Flk-1+ cells was further co-cultured with human AGM stromal cells in non-contact system.On co-culture days of 3,6,9 and 12 days,cells were collected for cell count,flow cytometry for Sca-1+c-kit+ cells analysis,and colony forming cell assay.RESULTS: During the EB formation,BMP4+VEGF promoted Flk-1+ cell genesis on day 9 at peak pencentage value of 27.53%±2.84％,which was statistically higher than that in control group as 8.77±1.10 (P<0.05).Collagenase-disassociated single cell from day 9 EB was co-cultured with human AGM stromal cells of hAGMS3 or hAGMS4 for further hematopoietic differentiation.On day 6 Sca-1+c-kit+ cells got to peak value as 7.31%±1.21％ ［(2.57±0.48) folds］ and 7.62%±1.52％ ［(2.35±0.36) folds］ in hAGMS3 and hAGMS4 feeder systems,respectively,both of which were greater than those values of no-stroma groups at the same culture duration (P<0.05).Colonogenic cell assay showed that these Sca-1+c-kit+ cells had ability of forming multiple lineage hematopoietic colonies.CONCLUSION: BMP4 in combination with VEGF promotes Flk-1+ cell genesis during EB formation in vitro.Stromal cells from early human embryonic AGM region further enhance the directed differentiation of these primitive cells into HSCs.This two-step induction differentiation model can be used for molecular mechanism study of ESCs hematopoietic differentiation.     

Effect of ex vivo short time expansion with SCF and IL-6 on the adhesion and transmigration activities of hematopoietic stem/progenitor cells

AIM:To investigate the expression and function of homing related molecules and transmigration ability of human cord blood CD34+ hematopoietic stem/progenitor cells after short time stimulation with cytokine SCF and IL-6.METHODS:CD34+ cells were separated by Ficoll density gradient centrifugation and stimulated by SCF and IL-6 cytokines for 48 h. The changes of CD49d (VLA-4), CD11a (LFA-1), CD62L (L-selectin) and CD184 (CXCR4) were analyzed by flow cytometry. The adherent and migration activities of CD34+ cells were evaluated in human fibronectin (FN) coated microplates (96 wells) and transwell system.RESULTS:The numbers of CD34+ cell expanded to 3 folds and the percentages of CD34+ cells that were positive expressions for CD49d, CD11a, CD62L or CD184 increased 1 to 2 folds after the cytokine stimulation. The spontaneous adhesion between CD34+, FN and SDF-1 induced migration increased after SCF+IL-6 stimulated.CONCLUSION:SCF+IL-6 can improve the most of the homing related characteristics and activities in the short time expansion of CD34+ hematopoietic stem/progenitor cells, which may be partly related to the increased intrinsic homing potential.     

Supportive and expansive effects of aorta-gonad-mesonephros (AGM)-derived stromal cells on hematopoietic stem in vitro

AIM: To explore the supportive and expansive effects of aorta-gonad-mesonephros(AGM) region derived stromal cells on hematopoietic stem cells (HSC) in vitro.METHODS: The murine stromal cells were separated and cultured from AGM region of a 11 day postcoitum (dpc) mouse embryo and 6 week mouse. After identification by Wrights staining and flow cytometry, the stromal cells were co-cultured with the embryonic stem cell(ESC)-derived, cytokine-induced HSCs, and the maintenance and expansion of HSCs were evaluated by detecting CD34+，CD34+Sca-1+cells with flow cytometry.Blast colony-forming cell (BL-CFC) and high proliferative potential colony-forming cells(HPP-CFC) were determined by semi-solid medium clonal culture.RESULTS: AGM-derived and bone marrow(BM)-derived stromal cells were similar in morphology and phenotype, and had common character of stromal cells. Supported by AGM stromal cells or by BM stromal cells, more primitive progenitor cells HPP-CFC were expanded, but BL-CFC expansion was only detected in AGM-derived stromal cells. In the supporting of BM stromal cells CD34+ hematopoietic stem/progenitor cells were expanded 3-4 times, but no significant expansion in CD34+Sca-1+ cells was observed. While in the supporting of AGM stromal cells, both CD34+ hematopoietic stem/progenitor cells and CD34+Sca-1+ cells were expanded significantly from 4 to 5 times, respectively (P<0.05).CONCLUSION: AGM-derived stromal cells significantly support the expansion of HSC, and also maintain the self-renewal activity and multi-lineage differentiation of HSC in vitro.     

Differentiation potential of human placenta derived mesenchymal stem cells into vascular endothelial cells

AIM: To investigate the differentiation potential of human placenta derived mesenchymal stem cells (PDMSCs) into endothelial cells (ECs) in vitro. METHODS: PDMSCs were isolated from human placenta tissues, characterized by flow cytometry and induced to differentiate into endothelial cells with 50 μg/L VEGF and 10 μg/L bFGF. To detect the specific markers of ECs during the process of differentiation, the method of immunocytochemistry was performed. The specific structure and function of endothelial cells were observed by transmission electron microscopy and in vitro angiogenesis assay kit, respectively. RESULTS: CD105 and CD106 were positive in PDMSCs, while CD34,CD45 and CD31 were negative.The ECs differentiated from PDMSCs showed cobblestone-like morphology, and expressed early endothelial marker of Flk-1/KDR and mature endothelial markers of CD31, vWF and CD144/VE-cadherin in a time-dependent manner during the endothelial cell differentiation (0 day, 4 days, 8 days and 12 days). The endothelial specific structure, Weibel-palade body, was observed under transmission electron microscope. The inoculation of ECs on the extra cellular matrix gel formed capillary-like structures. CONCLUSION: Plentiful PDMSCs can be isolated from placenta, and differentiate into the cells with functional characteristics of ECs in vitro, indicating that the placenta tissues will become optimal source of seed cells for vascular engineering and regenerative medicine.     

Isolation of endothelial progenitor cells from cord blood with CD133 immunomagnetic sorting

AIM: To isolate, purify and differentiate endothelial progenitor cells from cord blood in vitro and to study their biological characteristics. METHODS: CD133+ cells were selected from fresh cord blood mononuclear cells (MNC) by magnetic activated cell-sorting system (MACS). EPC was studied by flow cytometry, immunocytochemistry and immunofluorescence staining. Isolated cells were cultured in IMDM medium supplemented with or without VEGF, bFGF, SCF. RESULTS: The percentage of CD133+ cells of cord blood MNC was (1.41±1.14)%, and purity was 75%-85% (FACS method). CD133+ cells were grown on fibronectin-coated chamber slides in the presence of VEGF, bFGF, SCF. Within 1-2 hours of culture cells became adherent. On day 7-10, the adherent cells displayed a typical “cobblestone” morphology. After 14 days of culture, the adherent cells revealed a heterogeneous cell population, comprising small-sized round cells, spindle-like cells and formed tube-like structure. Weibel-Palade bodies were shown on the transmission electron microscopy photomicrographs. Compared with the original, cell markers CD133 and CD34 decreased significantly (77.0%±3.3% to 1.6%±2.2% and 93.1%±4.7% to 37.4%±4.9%, P<0.05), while Flk-1 increased significantly (from 22.3%±3.3% to 94.3%±4.1%, P<0.05) after 14 days of culture with VEGF, bFGF, SCF. The vWF was strongly expressed (77.9%±3.3%) on the 14th day later. CONCLUSION: Vascular endothelial progenitor cells were isolated from cord blood with specific expression of CD133/CD34/Flk-1. With the stimulation of the growth factors, seven-ten days after culture EPCs could be turned to endothelial cells.     

In vitro hematopoietic stem cells from embryonic stem cells reconstruct hematopoiesis in mice

AIM: To investigate the potential of hematopoietic stem cells (HSCs) derived from mouse embryonic stem cells (ESCs) to reconstruct hematopoiesis in vivo. METHODS: Using a three-step method, a mice embryonic stem cell line, E14.1 was induced into hematopoietic stem cells. The cell markers with CD34+/ Sca-1+ were identified by flow cytometry analysis, then HSCs (1×109 cells/L) from third-step were injected into SCID mice for observing teratoma formation. To validate function of HSCs, colonogenic cell assay was conducted and the hematopoiesis in lethally irradiated mice was reconstituted. RESULTS: The method of three-step differentiation, combined to use more hematopoietic stimulating factor promoted the E14.1 cell differentiation into HSCs with highest percent of CD34+/Sca-1+ cells (as high as 58.64%±4.20%) with more CFU-E, CFU-GM and CFU-GEMM populations. The cells showed the character of hematopoietic progenitors by Wright-Giemsa staining. Positive selected CD34+/Sca-1+ cells by magnetic sorting from third-step differentiation were transplanted into 7 lethally irradiated female mice while predominant hematopoietic reconstitution were observed in 10 d after transplantation, with 71.4% (5/7) successful engraftment rate. Three recipients showed that the cell population of the peripheral blood leukocytes, red blood cells and hemoglobin approached to normal index at 40 d after transplantation, but followed relative slow renew in platelet count. Survival rate in transplant group was 43%, compared to 100% mortality in control mice. Karyotyping assays confirmed the female mice with XY. CONCLUSION: The three-step differentiation and the culture conditions described here support the differentiation of mouse ESCs into HSCs. HSCs derived from mouse ESCs can reconstruct hematopoiesis.     

The ex vivo expansion characteristic of endothelial progenitor cells

AIM: To explore the ex vivo expansion characteristics of the endothelial progenitor cells (EPCs). METHODS: CD34⁺ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded at the same conditions as that for total MNC, coincubation of CD34⁺ and CD34^- from the same donation for EPCs. In addition, we tested the effect of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis. EPCs were determined and quantified by immunocytochemistry and flow cytometry. RESULTS: Coculture of CD34⁺ and CD34^-,total MNC led to a significant increase in the expansion of CD34⁺ cells compared with CD34 enrichment (P＜0.05). There was a trend toward decreased apoptosis in cultures when early passage was performed once the linear cord like structures appeared. There was no significant effect on apoptosis between with VEGF and without VEGF group (P＞0.05). These differentiated EPCs were stained positive for CD34⁺, von Willebrand factor (vWF), KDR, CD31 and incorporate acetylated low-density lipoprotein (LDL). CD34⁺ and AC133⁺cells accounted for 68.2%±6.3% (n=6) and 57.2%±9.8% (n=6) of attaching (AT) cells at day 7 of culture, respectively. CONCLUSIONS: Coculture of CD34⁺ and CD34^- or culture of MNC enhances ex vivo expansion of EPCs. Early passage decreases apoptosis rate, VEGF has no significant effect on ex vivo expansion of EPCs.     

Human bone marrow mesenchymal stem cells differentiate toward endothelial lineage in vitro

AIM：To investigate the cytological basis and differentiating conditions of human bone marrow mesenchymal stem cells(hMSCs) differentiated into cells of the endothelial lineage in vitro.METHODS：hMSCs were isolated by density gradient centrifugation and fractionated on a 1 073 g/L Percoll.The combination of VEGF165 and various matrix proteins including fibronectin (FN) and typeⅠ collagen (Col) was used to induce hMSCs in vitro.Cells were characterized by immunohistochemistry,cytochemistry,FACS and ultrastructure to identify and detect the differentiated population and markers.RESULTS：hMSCs was positive for KDR.PAS reaction was positive and ultrastructure of hMSCs showed glycogen-pool in ectoplasm.Glycogen reducing or disappear suggested that stem cells have occurred differentiation.Induction of hMSCs resulted in the increase of KDR,β1 integrin and CD34.CONCLUSION：hMSCs were induced to a transit population (TP) that differentiated toward the endothelial progenitor cells (EPC),but not a really EPC.hMSCs pedigree diagram of differentiation was hMSCs→TP→EPC→endothelial cells (ECs).     

Direct differentiation of embryonic stem cells into neural cells without embryonic body culture period in vitro

AIM:To investigate the feasibility and effect of directly differentiation of embryonic stem cells (ESC) into neural cells induced by retinoid acid (RA) without embryonic body (EB) culture period in vitro. METHODS:ESC were digested and divided into 4 groups:group A and B were undergone EB culturing. After that, cells in group A were induced by RA, cells in group B were differentiated spontaneously, cells in group C were committedly induced by RA directly without EB culturing, and cells in group D were differentiated spontaneously without EB period. Morphologic changes were observed under inverted microscope and scanning electron microscope. MAP-2 and GFAP were detected by immunocytochemistry and flow cytometry after differentiated for 9 days. RESULTS:In groups A or C, neuron-like cells increased gradually, forming neural network. At the 9th day, a large part of cells in these groups were MAP-2 positive cells, and the positive rate was higher than that in groups B or D (P<0.01). Groups B and D were almost epithelial-like cells. At the 9th day, GFAP positive cells were predominant. The rate of GFAP staining cells in groups A or C was significantly lower than that in groups B or D (P<0.01). There was no significant difference of MAP-2 or GFAP positive rates between group A and C, nor between group B and D (P>0.05). CONCLUSION:ESC was directly induced into neural cells by RA without EB culture period in vitro. This modified method has the same effect as the traditional RA 4－/4＋ assay and can replace the traditional method.      

Expression relevance of GATA-1 and miR-451a in erythroid differentiation of K562 cells under hypoxia

AIM To investigate the expression relevance of GATA binding protein-1 (GATA-1) and microR?NA-451a (miR-451a) in erythroid differentiation of human chronic myeloid leukemia K562 cells under hypoxia. METHODS The K562 cells were divided into 2 groups: normoxia group and hypoxia (1% O₂) group, and 40 μmol/L hemin chloride was used to induce K562 cell differentiation for 48 and 72 h. The mRNA expression of γ-globin was detected by RT-qPCR, hemoglobin production was observed by benzidine staining, and flow cytometry was used to detect CD235a expression for verifying erythroid differentiation model. The protein expression of GATA-1 during K562 cell differentiation under normoxia and hypoxia was determined by Western blot. RT-qPCR was used to detect the mRNA expression of GATA-1 and the expression level of miR-451a, and their correlation was analysis. The K562 cells were infected by lentivirus for over-expression or knock-down of GATA-1. Meanwhile, the morphological changes of the cells in the above groups were analyzed by Wright-Giemsa staining method to clarify the erythroid differentiation of K562 cells. The expression miR-451a was detected by RT-qPCR after GATA-1 over-expression or knock-down. REULTS: Under normoxia and hypoxia conditions, the expression levels of γ?-globin and CD235a and the positive rate of benzidine staining at 48 and 72 h were significantly higher than those at 0 h (P<0.05).At 72 h, the expression levels of γ?-globin and CD235a and the benzidine staining positive rate in hypoxia group were significantly higher than normoxia group (P<0.05). The expression of GATA-1 mRNA and miR-451a under hypoxia showed an upward trend during the erythroid differentiation of K562 cells, and was significantly higher than that in normoxia group at 72 h (P<0.05). Correlation analysis showed that the mRNA expression of GATA-1 was positively correlated with miR-451a expression under hypoxia (P<0.01). After over-expression of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the positive rate of benzidine staining, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly higher than those in negative control group (P<0.05). After knock-down of GATA-1 under hypoxia, the expression of γ-globin and CD235a, the benzidine staining positive rate, and the cell counts of size augmentation, nuclear deflection and nuclear shrinkage at 72 h were significantly lower than those in negative control group (P<0.05). Compared with negative control group under hypoxia, the expression of miR-451a was significantly increased after GATA-1 over-expression (P<0.05), while the expression of miR-451a was significantly decreased after GATA-1 knock-down (P<0.05). CONCLUSION Hypoxia increases the expression of GATA-1 and then up-regulates miR-451a to promote erythroid differentiation of K562 cells.     

Hemangioblastic characteristics of human adipose tissue-derived adult stem cells in vivo

AIM: To investigate whether Flk1+CD31-CD34- cells isolated from human adult adipose tissue have characteristics of hemangioblasts in vivo. METHODS: After sublethally irradiated (300cGy) with a caesium source, the female non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice were injected with human adipose tissue-derived Flk1+CD31-CD34- cells (105 cells per mouse) via tail vain with 0.4 mL Roswell Park Memorial Institute medium (RPMI-1640). The control mice received the same volume of RPMI-1640 medium. All mice were killed 2 months after transplantation for further study. The differentiation potential of Flk1+CD31-CD34- cells was assessed in bone marrow and gastrointestinal tract by the methods of flow cytometry, RT-PCR, FISH, and triple-color immunofluorescence. RESULTS: Flk1+CD31-CD34- human adipose tissue-derived adult stem cells differentiated into endothelial cells and hematopoietic cells at the single-cell level in vivo. CONCLUSION: Human adult adipose tissue-derived Flk1+CD31-CD34- cells bear characteristics of hemangioblast in vivo and may have potential application for the treatment of hematopoietic and vascular diseases.     

红掌愈伤组织增殖分化条件的优化

以红掌愈伤组织为材料,研究影响红掌愈伤组织增殖和分化以及芽增殖的因素。结果表明:以MS+6-BA1.0 mg/L+KT 0.3 mg/L+蔗糖30 g/L+琼脂8 g/L为红掌愈伤组织增殖和分化的最佳培养基,培养到8周绝对生长速度达到910.31%、分化率达到100%,到150 d时分化指数达到24.17;在芽增殖培养试验中,以1/2 MS+6-BA2.0 mg/L+NAA 0.5 mg/L+KT 0.1 mg/L+蔗糖30 g/L+琼脂8 g/L为适宜培养基。在培养方式上,液体培养优于固体培养。     

Effect of proliferating cell nuclear antigen specific antisense oligonucleotide on differentiation of cord blood CD34+ cells

AIM:To study the effect of proliferating cell nucler antigen antisense oligonucleotide on ex vivo expansion of cord blood CD34+ hematopoietic stem/progenitor cells. METHODS:CD34+ cells were purified from fresh cord blood by immunomagnetic beads. CD34+ cells were incubated in liquid culture system with different concentrations of PCNA-ASODN. Using flow cytometry, the number of different kinds of stem/progenitor cells and PCNA expression were measured after CD34+ cell incubation. RESULTS:PCNA was lowly expressed in low experiential group, with a positive rate of (27.2±3.6)% and (19.0±1.5)%, the positive rate of control group was (53.8±8.3)% (P<0.01), a high significant difference was observed. Just in low concentration of PCNA-ASODN, the percentage of CD34+cells were increased to (33.4±3.2)%, CD34+CD38-cells expanded (57.8±9.9) folds, and the percentage of CD34+cells were (25.2±2.6)% (P<0.01), the CD34+CD38-cells expanded (43.5±7.4) folds (P<0.05), it has significant difference compared with the control group. CONCLUSION:Low concentration of PCNA-ASODN decreases PCNA expression effectively, slows down differentiation of CD34+cells during ex vivo expansion procedure, and improves the expansion efficiency.     

Promoting effects of activated Nodal signal pathway on definitive endoderm induction from mouse embryonic stem cells

AIM: To study the process of promoting mouse embryonic stem cells (ESC) to specify to definitive endoderm by up-regulating of Nodal signal pathway in order to find the best cultivated systems of differentiation of mouse ESC to definitive endoderm cells. METHODS: The cells were divided into different groups based on the culture medium: ESC group (serum-free medium + LIF), natural differentiation group (serum-free medium) and activin A group (serum-free medium +50μg/L activin A). The cells and the sterilized coverslips with cells were collected at 1, 3, 5 and 7 d of the cultivation. The proportion of CXCR4⁺ cells was detected by flow cytometry. The expression of CXCR4 was determined by immunocytochemical method, and the protein expression of OCT4 and CXCR4 was detected by Western blot. RESULTS: The proportion of CXCR4⁺ cells showed no dramatic change in ESC group along with the extending of cultivation day, while there were gradually increased in natural differentiation group and activin A group and the highest level was observed at 5 d. Among the 3 groups, the proportion of CXCR4⁺ cells at 5 d was the highest in activin A group. The brown or tan staining in the cells observed under microscope was considered as positive CXCR4 by immunocytochemistry. The protein levels of OCT4 and CXCR4 in ESC group along with the extending of cultivation days was observed. The expression levels of OCT4 were gradually decreased in the cells in natural differentiation group and activin A group, while those of CXCR4 were gradually increased with the highest level at 5 d. It was highest in the cells in activin A group. CONCLUSION: The proportion of definitive endoderm was the highest at 5 d of the induction during in vitro mouse ESC differentiation. Up-regulation of Nodal signal pathway by adding activin A at the early stage of mouse ESC differentiation promotes mouse ESC to specify to definitive endoderm with CXCR4 molecular marker.     

Supportive effects of human AGM region and fetal liver stromal cells on differentiation of murine embryonic stem cells into hematopoietic stem cells and in vivo hematopoietic reconstitution

AIM: To investigate the differentiation of murine embryonic stem cells (ESCs) into hematopoietic stem cells (HSCs) by the supportive effects of human aorta-gonad-mesonephros (AGM) region and fetal liver (FL) stromal cells.METHODS: E14 ESCs were induced into embryoid body (EB) first. Then the cells from EB were further co-cultured with human AGM region and FL stromal cells in non-contact system. On day 6, the cells derived from EB were collected for Sca-1⁺c-Kit⁺ cell analysis by flow cytometry, colony forming unit (CFU) assay and teratoma formation checking. BALB/c female mice conditioned with lethal dose of γ-ray irradiation were transplanted with EB cells from different culture systems. The survival rates, engraftment of donor cells, reconstitution of hematopoietic were monitored.RESULTS: Sca-1⁺c-Kit⁺ cells in EB cells co-cultured with human AGM region and FL stromal cells had the value of (21.96±2.54)%, and the total CFU was as (520±52)/10⁵ cells, which were statistically greater than those in EB cells only cultured with human AGM region stromal cells (P＜0.05). No teratoma was found in NOD-SCID mice after subcutaneous injection of EB cells co-cultured with human AGM region and FL stromal cells. In BALB/c female mice transplanted of EB cells co-cultured with human AGM region and FL stromal cells, the survival rate was 77.8%, and the peripheral blood cell count was obviously improved on day 14. PCR results showed the recipients all had sry gene copies from donor in bone marrow. The recipient mice transplanted with EB cells only cultured with human AGM region stromal cells all died within 15 days.CONCLUSION: Stromal cells from human AGM region and FL enhance the directed differentiation of ESCs into HSCs which can reconstruct hematopoiesis in vivo.     

Comparison of different conditions inducing embryonic stem cells

AIM: To evaluate the different conditions inducing mouse embryonic stem cells (ESC) in vitro to differentiate into cardiomyocytes. METHODS: BRL conditioned medium was used to promote the growth of ESC and maintain them in an undifferentiated state. During the inducing process, retinoic acid (RA), DMSO, activin-A and TGF-β₁ were used as inducing reagents, and made up six kinds of differentiating medium. Then a three-step method inducing ESC cultured in hanging drops, in suspension and in plating was used to induce the differentiation of ESC. RESULTS: ESC were induced in vitro to differentiate into cardiomyocytes. Of all groups, the highest differentiating rate was observed in the group induced by activin-A (20 μg/L) and TGF-β₁ (2 μg/L). CONCLUSION: The inducing conditions including activin-A (20 μg/L) and TGF-β₁ (2 μg/L) is very valuable in inducing ESC differentiation into cardiomyocytes.     

Effects of gemcitabine on inhibiting proliferation and inducing apoptosis of CD34+CD38- myeloid leukemia stem cells

AIM：To investigate the effects of gemcitabine (GEM), a novel analog of deoxycytidine and nucleoside reductase inhibitor similar to cytarabine (Ara-C) in structure, on the proliferation and apoptosis of myeloid leukemic stem cells (LSCs), CD34+CD38-KG1a cells. METHODS：The expression of CD34 and CD38 on the surface of acute myeloid leukemia KG1a cells was detected by flow cytometry. The effects of GEM at various concentrations for 24 h and sustained medication for 14 d and 21 d on the proliferation and colony-forming ability of KG1a cells were analyzed by soft agar colony-forming experiment. The changes of the cell cycle of KG1a cells treated with various concentrations of GEM were tested by flow cytometry. The apoptosis of KG1a cells was determined by flow cytometry with the staining of Annexin V-FITC and propidium iodide (PI). RESULTS：The percentage of CD34+CD38- cells in acute myeloid leukemia KG1a cells was (98.02±0.72)%.Treatments with 0.05 mg/L and 0.1 mg/L GEM for 24 h were similar to saline control group in cell cycle distribution of the KG1a cells, whereas KG1a cells treated with 0.5 mg/L GEM for 24 h were arrested at G0/G1 phase. After treatment with 0.1 mg/L and 0.5 mg/L GEM for 24 h, the colony numbers at 14 d and 21 d were lower than that in saline control group. No difference of the colony numbers between the cells treated with normal saline and 0.05 mg/L GEM for 14 d and 21 d was observed. After sustained medication with 0.05 mg/L, 0.1 mg/L and 0.5 mg/L GEM and Ara-C for 14 d and 21 d, the colony numbers decreased as compared to saline control group. Treatment with 0.5 mg/L GEM for 24 h increased the apoptotic rate of KG1a cells compared with saline control group, while treatments with 0.05 mg/L and 0.1 mg/L GEM for 24 h were similar to saline control group in cell apoptosis. CONCLUSION：GEM inhibits the proliferation and colony-forming ability, arrests the cell cycle at G0/G1 phase and induces apoptosis of CD34+CD38- acute myeloid leukemia cells.     

Construction of mouse ESC line stably expressing Pdx1 by Tet-On system

AIM To construct the mouse embryonic stem cell （ESC） line with stable pancreatic and duodenal homeobox 1 （Pdx1） expression by Tet-On system, which may lay a foundation for further research on the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells. METHODS The Pdx1-overexpressing lentiviral vector with green fluorescent protein marker and puromycin resistance was constructed by Tet-On system and was used to infect the mouse ESC. The cells were divided into 3 groups： blank control group （ESC group）, empty lentivirus control group （PDX1^- ESC group） and Pdx1 lentivirus transfection group （PDX1⁺ ESC group）. Flow cytometry was used to detect the transfected cells after screening by doxycycline （DOX）. The function of Tet-On system and the expression of Pdx1 gene were detected. The transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were sorted by flow cytometry, and constructed ESC line with stable expression of Pdx1 and negative control ESC line were verified. RESULTS （1） The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group were 90.72% and 94.01% after screening by DOX, respectively. The positive rates of transfected cells in PDX1^- ESC group and PDX1⁺ ESC group was 97.84% and 98.13% after sorting by flow cytometry, respectively. （2） With DOX, green fluorescence was observed in PDX1^- ESC group and PDX1⁺ ESC group. The mRNA and protein expression of Pdx1 was significantly increased in PDX1⁺ ESC group （P<0.05）. Without DOX, no green fluorescence was observed in the cells of the 3 groups, and no significant difference in the mRNA and protein expression of Pdx1 was observed （P>0.05）. （3） After 3 months of cryopreservation, the cell lines still survived in resuscitation culture and were regulated by DOX. CONCLUSION Using Tet-On system, the mouse ESC line with inducible Pdx1 expression were successfully established and could be used as an effective cell model to research the differentiation of Pdx1⁺ definitive endoderm cells into pancreatic cells.     

Synergistic effect of decitabine and valproic acid on apoptosis and cell cycle arrest at G0/G1 phase in gastric cancer MGC-803 cells

AIM: To investigate the synergistic effect of decitabine (DCA) and valproic acid (VPA) on apoptosis and cell cycle arrest at G₀/G₁ phase in gastric cancer MGC-803 cells. METHODS: Gastric cancer MGC-803 cells were used in the study and divided into the following groups according to the treatment with different drugs for 72 h: DCA 1.5 μmol/L,DCA 3.0 μmol/L, VPA 1.5 mmol/L, DCA 1.5 μmol/L+VPA 1.5 mmol/L and DCA 3.0 μmol/L+VPA 1.5 mmol/L. The early and late apoptotic rates were detected by annexin V and PI staining. The cell cycle was also determined by flow cytometry. The relative nm23-H1 mRNA expression level was measured by real-time quantitative PCR. RESULTS: The apoptotic rates in VPA 1.5 mmol/L+DCA 1.5 μmol/L group (early: 33.58%±3.88%; late: 31.52%±4.20%) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (early: 42.61%±4.23%; late: 38.01%±3.86%), the percentages of the cells in G₀/G₁ phase in VPA 1.5 mmol/L+DCA 1.5 μmol/L group (61.55%±2.38%) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (66.75%±2.48%), and the relative nm23-H1 mRNA expression levels in VPA 1.5 mmol/L +DCA 1.5 μmol/L group (1.84±0.46) and VPA 1.5 mmol/L+DCA 3.0 μmol/L group (3.02±0.36) were all significantly higher than those in the corresponding concentrations of single drug treatment groups (P<0.01). CONCLUSION: Synergistic effect of VPA and DCA on apoptosis and cell cycle arrest in gastric cancer MGC-803 cells is possibly via inactivation of nm23-H1 gene expression.